CN1331749A - Method for producing ascorbic acid intermediates - Google Patents

Abstract

The present invention relates to non-fermentative methods for the production of ASA intermediates, KDG, DKG, and KLG and methods for the regeneration of co-factor. The invention provides genetically engineered host cells comprising heterologous nucleic acid encoding enzymes useful in the process.

Description

Method for producing ascorbic acid intermediate

field of invention

The present invention relates to engineered routes, particularly biocatalytic methods, for the production of ascorbic acid intermediates. Specifically, the present invention provides methods for producing ascorbic acid intermediates in non-fermentative systems.

Background of the invention

Discovery of L-ascorbic acid (vitamin C, ASA) as a vitamin and antioxidant useful in the pharmaceutical and food industries. The synthesis of ASA has received great attention over the years due to its sizeable market and very high value as a specialty performance chemical. The Reichstein-Grussner method, a chemical pathway from glucose to ASA, was first published in 1934 (Helv. Chim. Acta 17: 311-328). Lazarus et al. (1989, "Vitamin C: Biotransformation by Recombinant DNA Methods", Genetics and Molecular Biology of Industrial Microorganisms, Washington Society for Microbiology, edited by C.L. Hershberger) disclose a method for the production of an ASA intermediate, 2-ketone A biotransformation method for base-L-gulonic acid (2-KLG, KLG), an intermediate that can be chemically converted into ASA. This biotransformation of carbon sources to KLG involves many intermediates, and this enzymatic process is associated with cofactor-dependent reductase activity. Enzymatic cofactor regeneration involves the use of enzymes to regenerate cofactors such as NAD+→NADH or NADP+→NADPH at the expense of another substrate, which is then oxidized.

There is a continuing need for economically viable methods of producing ASA intermediates. In particular, where the methods involve the use of enzymatic activities requiring cofactors, it would be particularly desirable to have methods that provide for the regeneration of cofactors. The present invention fulfills this need.

Brief description of the invention

The present invention relates to the non-fermentative production of ASA intermediates, such as KDG, DKG and KLG, from carbon sources in a biocatalytic environment, referred to herein as biocatalytic reactor. The biocatalytic environment can include viable or nonviable host cells that contain at least one enzymatic activity capable of processing a carbon source into a desired intermediate. In one embodiment, the desired intermediate is purified from the bioreactor by electrodialysis prior to conversion to the desired end product. See Figure 2, which schematically illustrates the production of these intermediates and products.

The present invention also relates to a method for the non-fermentative production of an ASA intermediate wherein the desired cofactors are regenerated. The present invention is based in part on the discovery that catalytic amounts of cofactors can be regenerated in a non-fermentative or in vitro method of producing KLG from a carbon source. In one embodiment of the invention, the desired cofactor is purified from the bioreactor by nanofiltration and reused.

When KDG is the desired ASA intermediate, the bioreactor is supplied with a carbon source that is biocatalytically converted to KDG by at least one oxidation step. In this embodiment, the host cell may contain a mutation in a gene encoding an oxidase activity that specifically oxidizes KDG such that KDG is not further converted to other intermediates or products.

When DKG is the desired ASA intermediate, the bioreactor is supplied with a carbon source that is biocatalytically converted to DKG by at least one oxidation step. Depending on the host cell used, the host cell may contain mutations in genes encoding oxidative or reductive enzymatic activity such that DKG is not further converted to other intermediates.

When KLG is the desired ASA intermediate, the bioreactor is provided with a carbon source that is biocatalytically converted to KLG by at least one oxidation step and at least one reduction step. Depending on the host cell used, the host cell may contain mutations in genes encoding oxidative or reductive enzymatic activity such that KLG is not further converted to other intermediates. This method provides a means for cofactor regeneration when the cofactor is required for the oxidation step and the reduction step.

In one embodiment, the host cell is recombinant and contains at least one heterologous enzymatic activity. In one embodiment, the enzymatic activity is associated with the host cell membrane; in another embodiment, the enzymatic activity is in solution; in another embodiment, the enzymatic activity is soluble within the cell; in another In one embodiment, the enzymatic activity is immobilized. The method can be carried out batchwise or continuously. The host cell is preferably a member of the family Enterobacteriaceae, which in one embodiment is a Pantoea species, especially Pantoea citrea. For example, Pantoea citrea is available from the ATCC under ATCC Accession No. 39140.

Host cells can be lyophilized, permeabilized, or otherwise treated to reduce their viability or mutated to eliminate glucose utilization for cell growth or metabolism, so long as this enzymatic activity is available to convert the carbon source to the desired intermediate. These intermediates can also be processed into the final product erythorbic acid or ASA.

Thus, in one aspect, the present invention provides a method for producing DKG or KDG from a carbon source, comprising: enzymatically oxidizing the carbon source by at least one oxidative enzymatic activity to obtain DKG or KDG. In another embodiment, the method comprises oxidizing the carbon source by a first oxidative enzymatic activity to obtain a first oxidation product, and oxidizing said first oxidation product by a second oxidative enzymatic activity to obtain KDG. In one embodiment, the first oxidase activity is GDH activity and the second oxidase activity is GADH activity. The host cell may also contain a mutation in the naturally occurring nucleic acid encoding KDGDH activity so that the KDG is not further oxidized. The KDG can also be converted into erythorbate. Alternatively, the method may further comprise oxidizing KDG to obtain DKG through a third oxidation enzymatic activity.

For the production of KLG, if the carbon source is KDG or if the carbon source is converted into KDG, the method comprises enzymatic oxidation of the KDG by at least one oxidative enzymatic activity into an oxidation product; A step of enzymatic reduction of the oxidation product to 2-KLG. Alternatively, if DKG is the carbon source or if the carbon source is converted to DKG, DKG is converted to KLG by reductive enzymatic activity.

The invention provides a method for the non-fermentative production of 2-KLG from a carbon source, wherein the method comprises the following steps in any order: enzymatically oxidizing the carbon source to a monoxidized product by at least one oxidative enzymatic activity; and by At least one reductase activity enzymatically reduces the oxidation product to 2-KLG. In one embodiment, said oxidative enzymatic activity requires an oxidized form of an enzymatic cofactor, said reductive enzymatic activity requires a reduced form of said enzymatic cofactor, said oxidized form of said cofactor and said Said cofactor in reduced form is recycled between at least one oxidation step and at least one reduction step. In one embodiment, the oxidized form of the enzymatic cofactor is NADP+ and the reduced form of said enzymatic cofactor is NADPH. In another embodiment, said enzymatic cofactor is NAD+ in oxidized form and NADH in reduced form. Other cofactors useful in the methods of the invention include ATP, ADP, FAD and FMN.

In one illustrative embodiment disclosed herein, the method comprises the following steps in any order, which may be performed simultaneously and/or sequentially in the method: Enzymatic oxidation of a carbon source by a first oxidative enzymatic activity to first oxidation product; enzymatic oxidation of the first oxidation product to a second oxidation product by a second oxidation enzymatic activity; enzymatic oxidation of the second oxidation product to a third oxidation product by a third oxidation enzymatic activity an oxidation product; and enzymatic reduction of a third oxidation product to 2-KLG by a reductase activity. In one embodiment, at least one of said first, second and third oxidative enzymatic activities requires an oxidized form of an enzymatic cofactor, and said reductive enzymatic activity requires a reduced form of said enzyme promoting cofactor, and wherein said cofactor in said oxidized form and said cofactor in reduced form are recycled between at least one oxidation step and the reduction step.

In one embodiment, the method is carried out in an environment containing an organic solvent, in another embodiment the method is carried out in an environment containing a long polymer. In yet another embodiment, the method is performed in an environment containing salt and over a range of salt concentrations.

The invention also provides vectors and recombinant host cells containing enzymatic activity for use in methods of producing ASA intermediates. In one embodiment, the host cell comprises a heterologous nucleic acid encoding a GDH obtainable from species including Thermoacidophilus, Cryptococcus and Bacillus species, and/or a DKG reductase that can be Obtained from Corynebacterium or Erwinia.

Brief description of the drawings

Figure 1 illustrates an in vitro method in which NADP+ and NADPH are recycled between oxidation and reduction steps.

Figure 2 schematically shows a route to an ASA intermediate. Steps marked a are enzymatic; steps marked b are either enzymatic or chemical transformations. In this illustration, the enzyme that converts glucose (Glc) to GA is GDH activity; the oxidase that converts GA to KDG is GADH activity; the oxidase that converts KDG to DKG is KDGDH activity, and the oxidase that converts DKG to KLG Reductase is DKGR active.

Figure 3 depicts the reductase activity at pH 7 and 30°C in the presence of 0-40% methanol.

Figure 4 depicts the reductase activity at pH 7, 22°C in the presence of 0-50% ethanol.

Figure 5 depicts reductase activity at pH 7 in _the presence of NaCI, KCl, _CaCl2 , _K2SO4 or potassium phosphate (KPi). The initial velocity is measured within 1 minute.

Figure 6 depicts the remaining reductase activity after incubation at pH 7 and 45°C with and without up to 500 mM 2-KLG.

Figure 7 shows the spectrophotometric measurement of NADPH in an in vitro cofactor recycling reaction. Absorbance was measured at 340 nm. The initial absorbance reading before enzyme addition was 0.7. Additional aliquots of GDH were added at approximately 12 and 23 minutes.

Figure 8 shows the spectrophotometric measurement of NADPH in an in vitro cofactor recycling reaction. Absorbance was measured at 340 nm. Sufficient NaCl was added at about 5 minutes to achieve a final concentration of 0.5M.

Figure 9 shows the reductase Km and relative Vmax for 2,5-DKG in the presence of increasing amounts of 2-KLG.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS DEFINITIONS

The following abbreviations used herein apply to glucose (G); D-gluconic acid (GA); 2-keto-D-gluconic acid (2KDG); 2,5-diketo-D-gluconic acid (2 , 5DKG or DKG), 2-keto-L-gulonic acid (2KLG or KLG), L-iduronic acid (IA), ascorbic acid (ASA), glucose dehydrogenase (GDH), gluconate dehydrogenase Hydrogenase (GADH), 2,5-diketo-D-gluconate reductase (DKGR) and 2-keto-D-gluconate reductase (KDGDH).

As used herein, the term "non-fermentative" or "in vitro" refers to a biocatalytic process that utilizes the enzymatic activity of cells. These cells may be non-viable or viable and not grow significantly. These cells can be genetically altered to eliminate their consumption of glucose and/or any intermediates produced. The in vitro methods of the present invention include the use of cell membranes containing the enzymatic activity associated with the biocatalytic process, the use of permeabilized cells or lyophilized cells containing the enzymatic activity associated with the biocatalytic process and the use of any ASA intermediate as a carbon source The biocatalytic conversion of the host cell or host cell membrane or any form of fragment providing the necessary enzymatic activity, the ASA intermediates include, but are not limited to, GA, KDG, DKG and KLG. The cell may be a recombinant cell containing a heterologous nucleic acid encoding the desired enzymatic activity or a naturally occurring cell containing the desired enzymatic activity. The term "bioreactor" as used herein refers to the environment in which non-fermentative or in vitro processes are performed.

Many enzymes are only active in the presence of cofactors, such as NAD+ or NADP+. The term cofactor as used herein refers to a substrate that is secondary in nature to an enzymatic reaction, but is important for the enzymatic reaction. The term "cofactor" as used herein includes, but is not limited to, NAD+/NADH; NADP+/NADPH; ATP; ADP, FAD/ _FADH2 and FMN/ _FMNH2 . The phrase "regeneration of a cofactor" or "recycling of a cofactor" in an in vitro system refers to the phenomenon of successive oxidation and reduction of a desired cofactor by biocatalysis, so that the desired cofactor exists in a suitable form for enzymatic catalysis. In the present invention, regeneration of the cofactor provides an environment in which the reduced form of the cofactor is available to reductases and the oxidized form of the cofactor is available to oxidases. The present invention encompasses the regeneration of cofactors between any enzymatic oxidation step and any enzymatic reduction step in the biocatalytic pathway from a carbon source to an ASA intermediate such as KLG. The required cofactors can be present in catalytic amounts provided by the host cell environment, or can be provided exogenously in oxidized or reduced forms in stoichiometric amounts at the beginning of bioreactor processing.

The term carbon source as used herein includes suitable carbon sources commonly used by Enterobacteriaceae strains, such as 6-carbon sugars, including but not limited to glucose, gulose, sorbose, fructose, idose, galactose and mannose, which Either the D or L form; or a combination of 6-carbon sugars, such as sucrose; or 6-carbon sugar acids, including (but not limited to) 2-keto-L-gulonic acid, iduronic acid, glucose acid, 6-phosphogluconic acid, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid, 2-ketogluconic acid phosphate, 2,5-diketo-L- Gulonic acid, 2,3-L-diketogulonic acid, dehydroascorbic acid, erythorbic acid and D-mannonic acid or enzymatic derivatives of such acids, provided that the carbon source can be converted, for example ASA intermediate of KDG, DKG and KLG.

As used herein, "Enterobacteriaceae" refers to bacterial strains that are generally Gram-negative in character and are facultatively anaerobic. Preferred Enterobacteriaceae strains are those capable of producing 2,5-diketo-D-gluconate from a D-glucose solution. Enterobacteriaceae capable of producing 2,5-diketo-D-gluconate from a D-glucose solution include, for example, Erwinia, Enterobacter, Gluconobacter, and Pantoea. Intermediates from carbon sources to ASA in microbial carbohydrate pathways include, but are not limited to, GA, 2KDG, 2,5DKG, 5DKG, 2KLG, and IA. In the present invention, a preferred fermentation strain of Enterobacteriaceae is Pantoea spp., especially Pantoea citrea. Four stereoisomers of ascorbic acid are possible: L-ascorbic acid, D-arabinosoascorbic acid (erythorbic acid), which has vitamin C activity, L-arabinosoascorbic acid, and D-xylosoascorbic acid. As used herein, the term ASA intermediate includes any product in the pathway to produce ASA including, but not limited to, KDG, DKG and KLG.

As used herein, the term "recombinant" refers to a host cell that contains a nucleic acid that is not naturally present in an organism and/or that has an additional copy of a recombinantly introduced exogenous nucleic acid. As used herein, the term "heterologous" refers to a nucleic acid or amino acid sequence that does not occur naturally in a host cell. As used herein, the term "endogenous" refers to a nucleic acid that is naturally present in a host.

"Nucleic acid" as used herein refers to nucleotide or polynucleotide sequences and fragments or portions thereof, and also to DNA or RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing sense or antisense chain. "Amino acid" as used herein refers to a peptide or protein sequence or portion thereof.

As used herein, the term "mutation" refers to any change in a nucleic acid that renders the product of the nucleic acid inactive or eliminated. Examples of mutations include, but are not limited to, point mutations, frameshift mutations, and partial or total deletion of a gene encoding an enzymatic activity, such as oxidase activity or reductive activity. In one embodiment of the production of KLG thereby regenerating the cofactor disclosed herein, the nucleic acid encoding the activity of membrane-bound GDH is mutated, thereby rendering exogenous GDH inactive. In another embodiment, the production of intermediate KDG is optimized by inactivating 2-keto-D-gluconate dehydrogenase.

As used herein, the phrase "oxidase" refers to an enzyme or enzyme system that can catalyze the conversion of a substrate of a given oxidation state to a product having a higher oxidation state than the substrate. The phrase "reductase" refers to an enzyme or enzyme system that can catalyze the conversion of a substrate of a given oxidation state to a product having a lower oxidation state than the substrate. Oxidative enzymes involved in the biocatalysis of D-glucose to KLG include D-glucose dehydrogenase, D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase, among others. Reductases involved in the biocatalytic pathway of the ASA intermediate to KLG include, inter alia, 2,5-diketo-D-gluconate reductase (DKGR), 2-keto reductase (2-KR), and 5-keto base reductase (5-KR). These enzymes include those naturally produced by the host strain or those introduced by recombinant means. In one embodiment disclosed herein, the process is carried out in a Pantoea citrea host cell in which naturally occurring membrane-bound, non-NADP+ dependent GDH activity is abolished and cytosolic NADP+ dependent GDH is recombinantly introduced . In another embodiment, a heterologous nucleic acid encoding reductase activity is introduced into the host cell. In a preferred embodiment, the reductase activity is obtainable from a Corynebacterium species or Erwinia species. The term "pathway enzyme" as used herein refers to any enzyme involved in the biocatalytic conversion of a carbon source to an ASA intermediate such as KDG, DKG and KLG.

As used herein, the term "isolated" or "purified" refers to a nucleic acid or protein or peptide or cofactor that has been removed from at least one component with which it is naturally associated. In the present invention, an isolated nucleic acid may include a vector comprising the nucleic acid.

It is clear in the art that acidic derivatives of carbohydrates, if in dissolved state, or if in solid form, in the undissolved state from which they were prepared, can exist in various ionized states depending on their surrounding medium. Use of a term, such as iduronic acid, to refer to these molecules is intended to include all ionization states of the organic molecule. Thus, for example, "iduronate", its cyclized form "idurolide" and "iduronate" refer to the same organic moiety and are not intended to specify a particular ionization state or chemical form.

detail

The present invention relates to the biocatalytic production of ASA intermediates, such as KDG, DKG and KLG, from carbon sources in vitro or in a non-fermentative environment. Depending on the intermediates produced, the process may require the presence of enzymatic cofactors. In a preferred embodiment disclosed herein, the enzymatic cofactor is regenerated. Due to the cost of cofactors, it is highly advantageous to use an in vitro method that allows regeneration of catalytic amounts of cofactors provided by the host cell environment or exogenously. Non-fermentative production of ASA intermediates

The invention provides a method for producing an ASA intermediate. These intermediates can be further processed into ASA, ASA stereoisomers or other products such as erythorbic acid. In a preferred embodiment, KDG is the desired ASA intermediate produced, and the bioreactor is provided with viable or non-viable Pantoea citrate host cells, as described in Example II herein, in code 2 - A mutation in the activity of keto-D-gluconate dehydrogenase. In this embodiment, the carbon source is biocatalytically converted to KDG by two oxidation steps, see FIG. 2 . In this embodiment, there is no need to regenerate the cofactor.

When DKG is the desired ASA intermediate, the bioreactor is provided with viable or nonviable P. citrate host cells and a carbon source that is biocatalytically converted to DKG through three oxidation steps, see Figure 2 . In this embodiment, there is no need to regenerate the cofactor.

When KLG is the desired ASA intermediate, the bioreactor is provided with viable or non-viable Pantoea citrea host cells and a carbon source such as glucose that passes through three oxidation steps and one reduction step as shown in Figure 2 Biocatalytically converted to KLG. In this embodiment, the reductase activity may be encoded by a nucleic acid contained within the Pantoea citrea host cell, or provided exogenously. In this embodiment, the first oxidative enzymatic activity requires the oxidized form of the cofactor and the reductive enzymatic activity requires the reduced form of the cofactor. In a preferred embodiment disclosed herein, Pantoea citrate cells are modified to eliminate the naturally occurring GDH activity and will be available from Thermoacidophilus, Cryptococcus and Bacillus species and be specific for NADPH+ The introduction of heterologous GDH into Pantoea cells provides a cofactor recycling system that requires and regenerates a cofactor. This embodiment provides a method for cofactor regeneration, thus eliminating the cost of continuous addition of exogenous cofactors to bioreactors producing KLG in Pantoea cells. In this embodiment, the host cell also contains a nucleic acid encoding 2,5-DKG reductase activity, or the 2,5-DKG reductase is added exogenously to the bioreactor.

In another embodiment for the preparation of KLG, a bioreactor is filled with Pantoea citrea cells containing nucleic acid encoding membrane-bound GDH, suitable enzymes and cofactors, and added gluconic acid to be converted to DKG. The reaction mixture was then made anaerobic and glucose was added. The GDH converts glucose to GA and the reductase converts DKG to KLG while recycling the cofactor. When these reactions are complete, oxygen is added to convert GA to DKG, and the cycle continues. in vitro biocatalytic environment

A biocatalytic process for converting a carbon source to an ASA intermediate begins with a suitable carbon source utilized by Enterobacteriaceae strains, such as a 6-carbon sugar, including for example glucose, or a 6-carbon sugar acid, such as KDG. Other sources of metabolites include, but are not limited to, galactose, lactose, fructose, or their enzymatic derivatives. In addition to a suitable carbon source, the medium must contain suitable minerals, salts, cofactors, buffers and other components known to those skilled in the art to maintain the culture and facilitate the enzymatic pathways necessary to produce the desired end product. point. Preferred salts for use in the bioreactor are Na ⁺ , K ⁺ , NH ₄ ⁺ , SO ₄ ^-- and acetate. The cells are first grown, the carbon source used for growth is removed in a non-fermentative process, the pH is maintained between about pH 4 and about pH 9, and there is oxygen.

In this in vitro biocatalytic process, the carbon source and its metabolites pass through an enzymatic oxidation step or an enzymatic oxidation and an enzymatic reduction step, which can be performed outside the host cell intracellular environment and utilize enzymatic Activity, through a pathway to produce the desired ASA intermediate. These enzymatic steps can be performed sequentially or simultaneously in a bioreactor, some of which require cofactors in order to produce the desired ASA intermediate. The present invention encompasses an in vitro method in which host cells are treated with organic matter, as described in Example V, such that the cells are rendered non-viable, while the enzyme remains available to oxidize and reduce the desired carbon source while biocatalyzing the carbon source to an ASA intermediate and/or its metabolites. The invention also encompasses an in vitro method wherein host cells are lyophilized, permeabilized by any means, spray dried, disrupted or otherwise treated so that the enzymes can be used to convert the carbon source to the ASA intermediate.

The oxidative or reductive enzymatic activity can be bound to the host cell membrane, immobilized, for example, on a resin, such as AminoLink coupled gel (from Pierce Chemical Co), immobilized on a polymer, or dissolved in the bioreactor environment. In a preferred embodiment, at least one oxidase is membrane bound. The environment can be in an organic or aqueous system or a combination of both, and can be in one vessel or in multiple vessels. In one embodiment, the process is carried out in two vessels, one with oxygen and one without oxygen. For example, membrane-bound enzymes that require oxygen (GDH, GADH, KDGDH) can be separated from those that do not (cofactor-dependent GDH, cofactor-dependent DKGR), which allows the use of smaller volumes of aerobic vessels, thus Reduced costs. Bioreactors can be run batchwise or continuously. In a batch system, all broth is harvested at the same time, regardless of what is added. In continuous systems, broth is regularly removed for downstream processing while new substrate is added. The resulting intermediates can be recovered from the fermentation broth by various methods including ion exchange resins, absorption or ion blocking type resins, activated carbon, concentrated crystallization, passage through membranes, etc.

A bioreactor process may also involve more than one type of cell, for example one type of cell may contain oxidizing activity and a second type of cell may contain reducing activity. In another embodiment, the host cells are permeabilized or lyophilized (Izumi et al., J. Fermentation Technology, 61 (1983) 135-142), as long as the necessary enzymatic activity remains available for conversion of the carbon source or its derivatives. Bioreactors can be performed with some enzymatic activity provided from an exogenous source and in an environment that provides solvent or long polymers to stabilize or increase the enzymatic activity. In one embodiment disclosed herein, methanol or ethanol is used to increase reductase activity. In another embodiment, Gafquat is used to stabilize the reductase (see Gibson et al. US 5,240,843).

In one illustrative bioreactor described herein, the host cell is a permeabilized Pantoea citrea cell supplied with D-glucose as a carbon source, which is subjected to a series of oxidation steps by enzymatic conversion. To obtain KLG, oxidases include GDH, GADH and DGDH, and the reduction step involves 2 DKGR (see US 3,790,444). The KLG produced by the method of the invention can be further converted into ascorbic acid and KDG into erythorbic acid by means known to those skilled in the art, see for example Reichstein and Grussner, Helv.Chim.Acta., 17, 311-328 ( 1934). cofactor regeneration

One of the advantages of the method of the present invention resides in the regeneration of cofactors required for pathway enzymes. Examples of cofactors useful in the present methods include, but are not limited to, NAD+/NADH; NADP+/NADPH; ATP; ADP, FAD/ ^FADH2 and FMN/ ^FMNH2 .

In one embodiment of the invention, the carbon source is converted to KLG in a process involving regeneration of the cofactor, as shown in FIG. 1 . In this enzymatic cofactor regeneration process, 1 equivalent of D-glucose is oxidized to 1 equivalent of D-gluconic acid, and 1 equivalent of NADP+ is reduced to 1 equivalent of NADPH through the catalysis of GDH. Then 1 equivalent of D-gluconic acid produced by GDH is oxidized to 1 equivalent of 2-KDG, which is then converted into 1 equivalent of 2,5-DKG by the action of membrane-bound dehydrogenases GADH and KDGDH, respectively. The resulting 1 equivalent of 2,5-DKG is then reduced to 1 equivalent of 2-KLG, and this NADPH is reoxidized to 1 equivalent of NADP+ by the action of 2,5-DKG reductase, effectively recycling an equivalent of the cofactor , and thus can be used for the second equivalent of D-glucose oxidation. Other methods of cofactor regeneration may include chemical, photochemical and electrochemical means, wherein the equivalent of oxidized NADP+ is directly reduced to one equivalent of NADPH by chemical, photochemical or electrochemical means. The amount of cofactor added exogenously to the bioreactor is from about 1 [mu]M to about 5 mM, in a preferred embodiment from about 5 [mu]M to about 1 mM. As described in the examples herein, NaCl affects the Km of NADPH whereas KLG, the charged form, does not. So if there is NaCl in the bioreactor, then more NADPH will be required to maintain the optimum rate. Also, as disclosed in the examples, most of the salts tested had an effect on the thermostability of the reductase. It will be obvious to those skilled in the art to adjust the salt content according to the bioreactor conditions such as temperature so as to obtain a balance between thermal stability and acceptable speed. Cofactors added exogenously to the in vitro system may be added alone, or may be added together with other substrates involved in the biocatalytic conversion of the carbon source to the ASA intermediate. The method includes the use of cofactors immobilized on a support, chemically altered, such as attached to long polymers, and the use of cofactors in isolated or purified form.

Desired cofactors can also be purified from biocatalytic environments by nanofiltration and reused. The use of nanofiltration membranes for retaining cofactors is described eg in Seelbach et al. (1997, Enzyme and Microbial Technology, Vol. 20, pp. 389-392). Recombinant Methods Host Cells

Any oxidative or reductive enzymes required to direct the host cell's carbohydrate pathways to produce ASA intermediates, such as KDG, DKG or KLG, if not naturally present in the host cell, can be introduced by recombinant DNA techniques known to those skilled in the art. Alternatively, enzymes that block the desired pathway can be mutated by recombinant DNA methods. The invention includes recombinant introduction or mutation of any enzyme or intermediate required to achieve the desired pathway.

In one embodiment of the invention, a carbon source such as glucose is converted to KLG by multiple oxidation steps and one reduction step. In this embodiment, the first oxidation step and the reduction step require a cofactor. The host cell is Pantoea citrea, a naturally occurring nucleic acid encoding glucose dehydrogenase (GDH) is mutated such that the dehydrogenase activity is eliminated, and an exogenous GDH is introduced into the cell. The invention encompasses host cells with other mutations in enzymes in carbon flow pathways that affect production. For conventional techniques, see, for example, Maniatis et al. in "Molecular Cloning", a laboratory manual, Cold Spring Harbor Laboratory, New York, and Ausubel et al., "Current Techniques in Molecular Biology", 1989, Greene Publishing "Associates and Techniques described in Wiley Interscience, N.Y.

In one embodiment of the invention, a nucleic acid encoding DKG reductase (DKGR) is recombinantly introduced into a Pantoea fermenting strain. Many bacterial species have been found to contain DKGR, particularly members of the Corynebacterium genus, including Corynebacterium, Brevibacterium, and Arthrosporium. In one embodiment of the invention, 2,5-DKGR obtainable from a certain strain of Corynebacterium SHS752001 (Grindley et al., 1988, Applied and Environmental Microbiology 54:1770-1775) was recombinantly introduced into lemon In Pantoea. In another embodiment, the 2,5-DKG reductase obtained from Erwinia herbophila disclosed in US 5,008,193 to Anderson et al. is recombinantly introduced into Pantoea citrinum.

Sources of nucleic acids encoding oxidative or reductase enzymes include the following: Enzymes in Glucose Dehydrogenase Smith et al. 1989, J. Biochem.

261:973; Neijssel et al. 1989,

Antonie Van Leauvenhoek

56(1):51-61 Gluconate Dehydrogenase Matsushita et al. 1979, Biochem.

Journal 85: 1173; Kulbe et al. 1987,

       Ann.N.Y.Acad Sci 506:552 2-Keto-D-gluconate dehydrogenase Stroshane 1977 Biotechnol.

         BioEng 19(4) 4592-Ketogluconate Reductase "Journal of Microbial Genetics 1991,

                                                      

4,758,514; 5,008,193;

5,004,690; 5,032,514 vector sequences

The expression vectors for expressing these pathway enzymes of the present method, such as dehydrogenases or reductases, in the host microorganism contain at least one promoter associated with the enzyme, which promoter is functional in the host cell. In one embodiment of the invention, the promoter is the wild-type promoter of the selectase, in another embodiment of the invention, the promoter is heterologous to the enzyme, but in the host cell is still functional. In one embodiment of the invention, the nucleic acid encoding the enzyme is stably integrated into the genome of the microorganism.

In a preferred embodiment, the expression vector contains a multiple cloning site cassette, which preferably contains at least one restriction endonuclease site unique to the vector to facilitate nucleic acid manipulation. In a preferred embodiment, the vector also contains one or more selectable markers. As used herein, the term "selectable marker" refers to a gene capable of being expressed in a host microorganism to facilitate selection of those hosts containing the vector. Examples of such selectable markers include, but are not limited to, antibiotics such as erythromycin, actinomycin, chloramphenicol, and tetracycline.

A preferred plasmid for the recombinant introduction of non-naturally occurring enzymes or intermediates into Enterobacteriaceae strains is RSF1010, which is a mobile, but non-self-transmissive plasmid with a broad range of bacterial hosts, including Gram-negative and the ability to replicate in Gram-positive bacteria (Frey et al., 1989, "The Molecular Biology of IncQ Plasmids" in: Thomas (editor), "Promiscuous Plasmids in Gram-Negative Bacteria", Academic Press, London, pp. 79- 94 pages). Frey et al. (1992, Gene 113: 101-106) reported that three regions could affect the mobility of RSF1010. convert

Routine transformation procedures are taught in Current Techniques in Molecular Biology (Vol. 1, edited by Ausubel et al., John Wiley and Sons Co. 1987, Chapter 9), and include the calcium phosphate method, using diethylaminoethylglucose Transformation and electroporation of glycans. Various transformation procedures for introducing nucleic acids encoding pathway enzymes into a given host cell are known to those skilled in the art. The method includes pathway enzymes produced and purified from recombinant host cells and added exogenously to the in vitro environment in which the pathway enzymes are either heterologous or endogenous to the host cell expressed by the actively growing host cell or present in In the membranes of nonviable host cells. Pathway enzymes to be added exogenously can be produced recombinantly using a variety of host cells, including bacterial, fungal, mammalian, insect and plant cells. Plant transformation methods are taught in Rodriquez (WO95/14099, published May 26, 1995).

In a preferred embodiment of the method, the host cell is an Enterobacteriaceae. Included in the Enterobacteriaceae group are Erwinia, Enterobacter, Gluconobacter and Pantoea species. In the present invention, a preferred fermentation strain of Enterobacteriaceae is Pantoea spp., especially Pantoea citrea. In another preferred embodiment, the host cell is a Pantoea citrea containing pathway enzymes capable of converting a carbon source to KLG. The present invention includes pathways from a carbon source to KLG via any intermediate in a microbial carbohydrate pathway capable of producing KLG using a carbon source and via intermediates including, but not limited to, GA, 2KDG, 2,5DKG, 5DKG, and IA. In one embodiment, the nucleic acid encoding the pathway enzyme is introduced via a plasmid vector, while in another embodiment, the nucleic acid encoding the pathway enzyme is stably integrated into the host cell genome. Identification of transformants

Although whether a host cell has been transformed can be detected by the presence/absence of expression of a marker gene (which can suggest the presence or absence of a nucleic acid of interest), its expression should be confirmed. For example, if the nucleic acid encoding a pathway enzyme is inserted into a marker gene sequence, recombinant cells containing the insert can be identified by lack of marker gene function. Alternatively, marker genes can be placed in tandem with nucleic acids encoding pathway enzymes under the control of a single promoter. Expression of the marker gene in response to induction or selection is also usually indicative of enzyme expression.

Alternatively, host cells containing the coding sequence for a pathway enzyme and expressing the enzyme can be identified by various procedures known to those skilled in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques, which include membrane-, solution- or fragment-based techniques for the detection and/or quantification of nucleic acids or proteins.

Furthermore, the presence of an enzyme polynucleotide sequence in a host microorganism can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, parts or fragments of the enzyme polynucleotide sequence. Measurement conditions

Methods for detection of ASA intermediates, ASA and ASA stereoisomers include redox titration using 2,6-dichloroindophenol (Burton et al. 1979, J. Assoc. Pub. Analysts 17:105) or other suitable reagents, High performance liquid chromatography (HPLC) using anion exchange (J. Chrom. 1980, 196: 163) and an electro-redox step (Pachia, 1976, Analytical Chemistry 48: 364). Suitable controls for these detection methods will be readily apparent to those skilled in the art. Recovery of intermediates

Once produced, these ASA intermediates can be recovered and/or purified by any means known to those skilled in the art, including lyophilization, crystallization, spray drying, and electrodialysis, among others. Electrodialysis for the purification of ASA and ASA intermediates such as KLG is described, for example, in US5747306, issued May 5, 1998, and US4767870, issued August 30, 1998. Alternatively, these intermediates can also be formulated directly from the bioreactor and pelletized or made into a liquid formulation.

Those skilled in the art can more fully understand the manner and method for carrying out the present invention by referring to the following examples, which are not intended to limit the scope of the invention or the associated claims in any way. All literature and patent publications referred to herein are hereby incorporated by reference.

Example

Example I

This example describes a method for producing a Pantoea citrea host cell with a mutation in the naturally occurring GDH. Cloning of the glucose dehydrogenase gene (GDH) from Pantoea citrea: The glucose dehydrogenase gene was cloned by polymerase chain reaction (PCR). Two primers were used in this PCR: 5'AGGGAGTGCTTACTACCTTTATCTGCGGTATA3' and 5'CGCTAGCTGTGCAATCCATTGATTTTGCACA3'. After PCR, a DNA product of about 2 kb, pGEM-T (Promega), was cloned in the vector, and a recombinant E. coli with the correct DNA insert was identified, and the clone was named pRL. The DNA insert was analyzed by DNA sequencing and found to be 60-70% identical to the DNA sequence of the published GDH of Pantoea citrea strains.

A deleted GDH gene was generated by insertion of the chloramphenicol resistance gene:

In order to generate deletion mutants of the GDH gene in Pantoea citrea, first a recombinant copy of the gene to be deleted was generated by introducing a selectable marker, the chloramphenicol resistance gene (CAT). This in vitro produced copy was introduced into P. citrea and recombined with the wild-type copy by homologous recombination. The pRL DNA was then analyzed by digestion with various restriction enzymes. It was found that there are two Smal cleavage sites separated by about 700bp in the GDH encoding DNA. The pRL was digested with Smal to remove the 700bp fragment, which was then replaced with a 1.05kb DNA digested with a Smal containing the chloramphenicol resistance gene to generate a recombinant plasmid, which was named pRLcm4. This method for generating pRLcm4 is a standard technique used by those skilled in the art. The GDH-CAT coding sequence from pRLcm4 was further transferred to a plasmid pGP704. The DNA encoding the GDH-CAT cassette was removed from pRLcm4 by combined digestion with the restriction enzymes Aatll and Spel. Sticky ends of digested DNA were removed by treatment with T4 DNA in the presence of deoxynucleotide triphosphate mixture. This GDH-CAT cassette was then ligated to EcoRV digested pGP704. A recombinant plasmid of pGP704 containing the GDH-CAT cassette was identified and named p704RLcm.

Introduction of the deleted GDH gene into the chromosome of P. citrea: The plasmid p704RLcm was introduced into wild-type P. citrea by electroporation. First, the transformed cells were plated on an agar plate containing 12.5 μg/ml chloramphenicol, and resistant clones were observed. To differentiate true deletion mutants (which should exhibit a chloramphenicol-resistant phenotype) from cells solely harboring the plasmid p704RLcm, chloramphenicol-resistant colonies were selected against ampicillin, another antibiotic resistance marker vector for p704RLcm. Identification of ampicillin-sensitive clones. Several clones with normal phenotypes (chloramphenicol resistance and ampicillin sensitivity) were identified by biochemical assays, all clones exhibited a GDH negative phenotype. Southern blot analysis also confirmed that the wild-type GDH gene was replaced by the deleted copy.

Example II

Example II describes methods for producing host cells with a mutation in the naturally occurring 2-keto-D-gluconate dehydrogenase (E3).

According to the method of Mclntire et al. (Mclntire, W, Singer, T.P., Ameyama, M., Adachi, O., Matsushita, K. and Shinagawa, E. "Journal of Biochemistry" (1985) 231, 651-654) and The reference therein purifies 2-keto-D-gluconate dehydrogenase (EC 1.1.99.4) from Gluconobacter niger. Digestion of the purified protein with trypsin and chymotrypsin or other proteases produces peptide fragments, which are separated by HPLC or other techniques. Individual peptides were collected and sequenced. From these sequences DNA probes are synthesized which will anneal to the corresponding sequences in the genome of the host organism or a related organism. Larger fragments of the desired gene are amplified, purified and sequenced using standard PCR techniques. These fragments are used to hybridize to the gene, allowing the entire gene to be cloned and sequenced. Once the sequence was known, the gene was deleted as described in Example 1 for D-gluconate dehydrogenase (GDH).

Other methods of reducing or eliminating 2-keto-D-gluconate dehydrogenase include inhibitors (organic acids such as citric acid and succinic acid have been reported to inhibit 2-keto-D-gluconate dehydrogenase; Shinagawa, E. and Ameyama, M. Methods in Enzymology (1982) 89, 194-198), and changes in pH or temperature.

The enzyme activity or loss of activity can be determined using the assay described in Shinagawa and Ameyama.

Example III

Example III describes a method for the production of KLG in a bioreactor that regenerates the cofactor. Materials and Methods Cell Permeabilization

400ml of Pantoea citrea with a mutation in the naturally occurring membrane-bound GDH was grown to 80 OD (600nm) in 10g/L gluconate and mixed with 16ml of 10% toluene and 90% acetone at 22°C. The mixture was mixed for 3 minutes. The permeabilized cells were then centrifuged at 9000 rpm for 10 minutes, and the resulting cell pellet was washed with 400 ml of 50 mM tris, pH 7. Repeat the wash 2 more times to ensure removal of residual organic solvent. Reactor charge

The 400 ml permeabilized cells from above in 50 mM tris, pH 7 were placed in a 1 liter glass vessel equipped with stirrer, temperature controller, oxygen delivery tube, base delivery tube, sample port and oxygen and pH probes. Add 200 μl of MAZU defoamer (BASF) to the solution to control excessive foaming, feed compressed air into the container to make the temperature 28°C, turn on the stirrer and rotate it at 1200rpm until the oxygen probe reading exceeds 60% saturation. Then 16 g of crystalline glucose and 4 g of crystalline sodium gluconate were added to make a final concentration of 10 g/L gluconate and 40 g/L glucose. The mixture was reacted until all gluconate was converted to DKG. Keep glucose levels above 20g/L. As a result of cell permeabilization, minimal amounts of glucose enter non-productive cellular metabolism. The pH was maintained at 7 by controlled addition of 50% NaOH throughout. Addition of soluble enzymes and cofactors

Once gluconate was converted to DKG, 2000 units each of cofactor-dependent GDH and DKG reductase (for DKGR, 1 unit equals 1 OD absorbance change per minute when measured at 340 nm) and 400 μM NADP+ were added. The reactor was stirred, air fed, and maintained at 28°C as described above. Glucose was added periodically throughout the reaction to ensure a constant substrate supply for the two cofactor-dependent enzymes. result

Bioreactor experiments were performed with non-purified reductase A: F22Y/A272G (US 5,795,761 ) as a crude extract from E. coli. Acidophilus GDH and NADP+ were purchased from Sigma in purified form. The conversion rate of GA to DKG is greater than 10g/L/hr. The initial rate of formation of 2KLG is greater than 10 g/L/hr. The accumulation rate in the first 6 hours is above 5g/L/hr. The cofactor appeared to be stable during the first 6 hours and was mainly in the reduced form. The total turnover number was 537 (215mM 2KLG/0.4mM NADP+). The intermediates GA and DKG never exceeded 4 g/L during the first 6 hours. The run was stopped 6.5 hours after the start of the cell addition and a gradually reduced low agitation phase was carried out overnight at 22°C with a final titer of about 42 g/L of KLG.

Aliquots were removed during bioreactor incubation. The aliquots were first spun in a microcentrifuge to pellet the cells. For determination of remaining reductase activity, 25 μl of sample supernatant was added to a solution consisting of 910 μl buffer (50 mM bis-tris, pH 7), 20 μl DKG (70 mg/ml) and 250 μM N ADPH. Reductase activity was determined by monitoring the loss of absorbance at 340 nm over 1 min. GDH activity was measured as follows: Add 25 μl sample to a solution containing 520 μl buffer, 150 μl NaCl (1M), 200 μl urea (8M), 50 μl glc (1M) and 60 μl NADP+ (5 mM) and monitor the absorbance at 340 nm within 1 min increase. Both reductase and GDH showed full activity throughout the course of the bioreactor experiments.

Example IV

This example describes the production of KDG in an in vitro bioreactor.

Cells containing membrane-bound D-glucose dehydrogenase and D-gluconate dehydrogenase activity but not 2-keto-D-gluconate dehydrogenase activity were grown and harvested. An example of such a cell is Pantoea citrate, which has a mutation in 2-keto-D-gluconate dehydrogenase, and was grown and treated as in Example III. These cells were permeabilized as described in Example III. Glucose (crystal or solution) was added in aliquots or continuously. Its pH was maintained by the controlled addition of concentrated NaOH solution. This glucose is converted to D-gluconate and then to KDG. Product formation was monitored by analyzing aliquots on a suitable HPLC system. The product is recovered by centrifugation and concentration or removing the remaining liquid to remove the cells.

Example V

This example describes the addition of organic solvents to increase reductase activity.

Add 1-2 mg of DKG, 250 μM NADPH, F22Y/A272G reductase A and sufficient 50 mM bis-tris buffer, pH 7, to make a final volume of 1 ml into a cuvette. Reductase activity was determined by monitoring the increase in absorbance at 340 nm. The amount of reductase added typically results in a change in absorbance of 0.1-0.2 OD/min at room temperature or 30°C. Under the same conditions, an aliquot of methanol or ethanol was added to this solution, and the reductase activity was measured. The reductase activity at 30°C in the presence of different amounts of methanol is shown in Figure 3 and in Figure 4 the activity in the presence of ethanol at 22°C.

As shown in these figures, the reductase activity increased in the presence of a certain amount of methanol or ethanol. The optimum concentration range is 10-25% organic solvent.

GDH from Acidothermic bacteria shows a slight decrease in activity when incubated with 10% methanol (assay conditions 50mM Tris, pH 7, 12.5mM D-glucose, 250μM NADP+ in 1ml. Activity monitored by increase in absorbance at 340nm) . Permeabilized cells were incubated with 15% methanol and gluconic acid. The addition of methanol had no significant effect on the activity of D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase as monitored by product formation (HPLC analysis).

Addition of methanol or ethanol to a complete bioreactor reaction will increase reductase activity. Loss of GDH activity or other components can be overcome by adding more GDH or cells.

Example VI

Example VI describes reductase activity in the presence of Gafquat and PEG8000.

Incubate at 30°C with 250μM NADPH, 1-2mg/ml DKG and 0, 0.7% and 2.8% Gafquat (ISP Technologies, Inc.) or 0.5% PEG8000 in 1ml (50mM bis-tris buffer, pH 7) enzyme. Reductase activity was determined as in Example VI. As shown in Table 1, the addition of Gafquat increased the reductase activity by 80% compared to the activity without Gafquat. PEG8000 increased reductase activity by about 15%. % of polymer added to final solution % activity without additives Gafquat 0.7-2.8 180PEG8000 0.5 115 Table 1. Increase in reductase activity in the presence of Gafquat or PEG8000.

Example VII

Example VII describes reductase activity in the presence of salt.

The activity of reductase A F22Y/A272G was determined in the presence of different amounts of different salts. The assay is performed by adding reductase to a buffer containing 250 μM NADPH, DKG (1-1.5 mg/ml), 50 mM bis-tris, pH _7.0 and varying amounts of potassium phosphate, NaCl, KCl, _K2SO4 or _CaCl2 solution (final volume 1 ml). All reactions were performed at 30°C. These results are shown in FIG. 5 .

As shown in Figure 5, the reductase activity remained the same or slightly increased when incubated with up to 100 mM NaCl or KCl. When the salt concentration was increased to 250 mM, the activity decreased. Reductase activity decreased at concentrations of _CaCl2 or potassium phosphate of 20 mM or higher.

The reductase binding constant (Km) of NADPH in the presence of 200 mM NaCl was determined using standard biochemical techniques (Fersht, A. "Structure and Mechanism of Enzymes" (1977) W.H. Freeman and Company). These reactions were performed at 30°C in pH 7 bis-tris buffer containing approximately 1.5 mg/ml DKG and varying amounts of NADPH. The Km of NADPH was found to be 10-40 times higher in the presence of 200 mM NaCl than the Km determined in the absence of NaCl. The maximum velocity (Vmax) in salt was similar or slightly higher than Vmax without salt. One way to reduce the effect of salt on reductase activity is to increase the concentration of NADPH until it is Km or above under these conditions. Alternatively, the charged form containing KLG can be removed.

Example VIII

Example VIII describes the stability of reductase A F22Y/A272G in the presence of salt/product.

The thermostability of the reductase is greatly increased in the presence of salt. Reductase was measured in one of the following ways. In the first case, the reductase was added to buffer (50 mM bis-tris, pH 7) with or without different amounts of 2-KLG (0-500 mM). These solutions were then aliquoted (40 μl) into 1.5 ml microcentrifuge tubes. The tubes were then placed in a 45°C water bath and removed at set intervals. Residual reductase activity was then determined using a standard reductase activity assay. The results are shown in FIG. 6 .

As shown in Figure 6, there was no appreciable loss of any activity of the reductase in the presence of 500 mM 2-KLG under these conditions. However, the reductase incubated with buffer alone lost nearly half of its activity after 10 minutes. The intermediate 2-KLG concentration was partially stabilized.

at pH 7 and 45 °C in the presence of buffer (50 mM bis-tris, pH 7 or 25 mM MOPS, pH 7), 0.5 M NaCl, 0.5 M KCl, 0.5 M NH ₄ Cl, 0.5 M K ₂ SO ₄ and 0.1 M NaCl Incubation for reductase. As shown in Table 2 below, there was almost no loss of activity in the presence of these compounds, whereas the activity of the buffer-only reductase was nearly half lost. Apparently these compounds stabilize the reductase. Lower or higher levels of these compounds should also stabilize the reductase. Residual activity after 10-minute incubation of reductase samples after 12-minute incubation

Activity % Property % Buffer 25-400.5M NaCl 1000.5M KCl 1000.5M NH ₄ Cl 1000.5M K ₂ SO ₄ 1000.1M NaI 80-85100mM NADPH 80-90200mM K ₂ PO ₄ 65-78100mM K ₂ SO ₄ 20-1 . Reductase activity after incubation at room temperature or 45°C for 10 minutes. Activity is determined using standard assays.

The stability of 2-KLG and NaCl was compared. Temperature incubations were performed at 45.4°C in 25 mM MOPS, pH 7. A concentration of 20 mM 2-KLG or 20 mM NaCl was used. Assays were performed at 0, 5 and 10 minute time points. The results are listed in Table 3 below. As shown in Table 3, the same amount of NaCl stabilized the reductase better than 2-KLG. condition % activity, 0 minutes % activity, 5 minutes % activity, 10 minutes 20mM NaCl 100 72 59 20mM 2-KLG 100 51 29 All % in Table 3 are +/-10%.

The half-life of the reductase has been determined at 0-400 mM NaKLG at 46.6-46.9°C. This temperature was chosen in order to determine all half-lives at the same temperature. The buffer was 25 mM MOPS, pH 7.0. Aliquots were removed and assayed for remaining activity. Thermostability half-life assays were performed as follows: 450 [mu]l samples containing buffer, reductase and 2KLG (as used here) were placed in a microcentrifuge tube and heated in a water bath. Eight or nine aliquots were taken over the course of time from 10-30 minutes. Each aliquot was kept on ice and assayed in duplicate at the end of the experiment. Time is plotted against remaining activity. _Kf was determined by fitting the line using a computer graphics program for exponential decay. This value was then used to calculate the half-life (Fersht, A. "Structure and Mechanism of Enzymes" (1977) WH Freeman and Co.). The results are shown in Table 4 below. NaKLG concentration 0mM 100mM 200mM 300mM 400mM Half-life (minutes) 3.5+/-0.5 5.5+/-1 7.5+/-1.5 10+/-3 18.5+/-3

As the results in Table 4 show, NaKLG apparently stabilized the reductase, and this stabilization was concentration-dependent.

This data demonstrates that increasing amounts of salt can stabilize the reductase. Suitable salts for use in the bioreactor include ammonium sulfate, sodium acetate, ammonium acetate, ammonium chloride, sodium sulfate, potassium phosphate, sodium phosphate, sodium chloride, KCl, _NH4Cl , _{K2SO4 ,} and NaI. Those skilled in the art will realize that the optimum range of salt will be temperature dependent. Thus, in the bioreactor, either the temperature, or the salt concentration, or both can be varied to achieve the desired stability of the reductase. As shown in Table 4, at the lower temperatures typical of bioreactor operations, less salt must be used to provide the same amount of reductase stabilization.

Example IX

Example IX describes a method for determining the NADPH/NADP+ ratio and reaction equilibrium.

The reduced cofactor (NADPH) has a strong absorbance at 340 nm, while the oxidized cofactor (NADP+) does not absorb at this wavelength. Thus, the amount of NADPH present can be determined by the absorbance at 340 nm if the two cofactors are mixed together. The ratio of these two cofactors can be easily determined if the amount of NADP+ initially added is also known. This method can be used to determine how the addition of various components to a reaction, such as a cofactor recycling reaction, affects the reaction equilibrium.

Set up a 1 ml reaction in a cuvette at room temperature. The reaction consisted of buffer (50 mM bis-tris, pH 7), 5 mg glucose, 5 mg 2,5-DKG, 100 μM NADPH, 100 μM NADP+, reductase and glucose dehydrogenase (GDH). These enzymes were added last, the reaction was started, and cofactor levels were monitored at 340nm. After equilibrium was reached (Figure 7), additional GDH aliquots were added. Soon the balance shifts, favoring the emergence of more NADPH. Adding more GDH gave the same reaction.

Another 1 ml incubation reaction was set up as above. After reaching equilibrium, 29 mg of NaCl was added to make a final concentration of 0.5M NaCl. As shown in Figure 8, this dramatically shifts the balance in favor of NADPH.

Example X

Example X describes a cofactor recycling reaction.

The cofactor recycling reaction is carried out by adding reductase, GDH, glucose, 2,5-DKG and NADP+ into the reaction vessel. In addition, purified 2-KLG was added to some reactions, which were evaluated in the presence of product. These reactions are maintained to produce gluconic acid and 2-KLG. Cofactors are recycled between NADP+ and NADPH through the action of these two enzymes. Aliquots were removed periodically and analyzed by HPLC for the presence of substrate and product. The reaction was maintained at room temperature for at least 20 hours.

Make the reaction as small as 3ml. In the reaction, reductase, GDH, 10 mg/ml glucose and 10 mg/ml lyophilized 2,5-DKG were added to 50 mM bis-tris buffer. 2-KLG was added to some incubation reactions at a concentration of 75 mg/ml. Reactions were started by adding NADP+ (400 μM) at room temperature. The pH of the solution was maintained at 6-7.5 by adding a small amount of NaOH. Aliquots of glucose and 2,5-DKG were added periodically. At the end of the day, the reaction was left overnight at 4°C. It was warmed to room temperature the next morning, its pH was adjusted, and the reaction was continued. A small aliquot was removed and injected onto the HPLC. By comparison with a standard, the amount of gluconic acid and 2-KLG produced can be calculated. In a typical reaction, at least 60% of the glucose is converted to gluconic acid and at least 60% of the 2,5-DKG is converted to 2-KLG.

Example XI

Example XI describes NaCl kinetics.

When NaCl (100 mM or higher) was added to a standard reductase assay containing 250 μM NADPH and 10-20 mM DKG, the reductase rate decreased. By performing a kinetic analysis, it was found that sodium chloride increases the Km of the reductase of the cofactor NADPH. Km increases when more NaCl is added. The reductase appears to be inhibited by NaCl if less than fully saturating amounts of NADPH are used. To counteract this effect, more NADPH was added to the reaction so that it was several times the Km. This mode of inhibition is apparently competitive.

The Km of DKG in the presence of NaCl was determined using standard techniques. The concentration of NADPH used for each assay was adjusted such that the Km was at least 3-fold at each NaCl concentration. [NaCl], mM Km(mM) of NADPH Km(mM) of DKG 0 4-9 6-14 100 30-100 6-14 200 130-230 6-14 400 260-360 6-14 Example XII

Example XII shows 2-KLG kinetics.

The Km of DKG in the presence of 2-KLG is determined using standard biochemical techniques. The amount of 2-KLG was varied from 0 to 150 mM in pH 7 buffer. The Km of DKG at pH 7 is 10-12 mM. The Km of 2,5-DKG decreased as the concentration of 2-KLG increased. For example, the Km of DKG is 2-4 mM at 150 mM 2-KLG. The reaction speed also decreased, and the reaction speed decreased by 2-4 times when the concentration of KLG was increased from 0 to 150 mM. This behavior is consistent with noncompetitive inhibition.

The Km of NADPH with 100 mM 2-KLG was determined to be 4-9 mM. This was performed using standard biochemical techniques at pH 7, with a concentration of 2,5-DKG greater than 14 mM, and with a NADPH concentration that brackets the Km value.

Therefore, the presence of increased amounts of KLG in the bioreactor environment is expected to reduce reductase activity and slow down the overall reaction rate. Two ways to overcome this phenomenon are to recover KLG, for example by electrodialysis or by adding more reductase to the bioreactor.

Example XIII

Example XIII describes the synthesis of 2,5-DKG.

Pantoa citrate cells were incubated with 150 mM sodium gluconate in the appropriate buffer: 25 mM bis-tris or 25 mM MOPS at pH 6 was used. 25 ml of cells, buffer and substrate were added to a 125 ml baffled Erlenmeyer flask and incubated at 28°C with shaking at about 250 rpm. After 16-24 hours, the 2,5-DKG formed in the flask was monitored by HPLC analysis and activity assay. The cells were spun down and the supernatant removed. Materials are sterile filtered and they can be stored at 4°C or frozen. Alternatively, the material can be lyophilized to a solid.

Claims (62)

1. the method by non-fermentative production KDG of carbon source or DKG comprises: obtain KDG or DKG by this carbon source of at least one oxidation enzymatic organized enzyme enzymatic oxidation.

2. the process of claim 1 wherein that described KDG further changes into erythorbate.

3. the method for claim 1 comprises by first oxidation enzymatic activity the carbon source oxidation is obtained first oxidation products, and by second oxidation enzymatic activity described first oxidation products oxidation is obtained KDG.

4. the method for claim 3, wherein said first oxidation enzymatic activity are that active and described second the oxidation enzymatic activity of a GDH is a GADH activity.

5. the method for claim 1, this method is carried out in containing the environment of host cell.

6. the method for claim 5, wherein said host cell is nonviable.

7. the method for claim 5, wherein said host cell is for living.

8. the method for claim 5, wherein at least one oxydase combines with described host cell membrane.

9. the process of claim 1 wherein that at least one oxidation enzymatic activity is in dissolved state.

10. the method for claim 8, wherein said host cell contains a sudden change in the active naturally occurring nucleic acid of encoded K DGDH.

11. the method for claim 5, wherein said host cell are the member of enterobacteriaceae.

12. the method for claim 11, wherein said member is a general Pseudomonas bacterial classification.

13. the process of claim 1 wherein that at least a oxidation enzymatic activity is immobilized.

14. the method for claim 3 also comprises the following steps: by at least a oxydase the KDG enzymatic oxygen to be changed into an oxidation products; With by at least a reductase enzyme described oxidation products enzymatic reduction is become 2-KLG.

15. the method by the non-fermentative production 2-KLG of carbon source comprises the following steps of random order: by at least one oxidation enzymatic activity the carbon source enzymatic oxygen is changed into an oxidation products; With by at least a reductase enzyme described oxidation products enzymatic reduction is become 2-KLG.

16. the method for claim 15, wherein said carbon source are KDG.

17. the method for claim 15, wherein said oxidation enzymatic activity needs the enzymatic cofactor of oxidised form, described reduction enzymatic activity need reduce the described enzymatic cofactor of form and the recirculation between at least one oxidation step and at least one reduction step of the described cofactor of the described cofactor of wherein said oxidised form and described reduction form.

18. the method for claim 15 comprises the following steps of random order:

A. by first oxidation enzymatic activity the carbon source enzymatic oxygen is changed into first oxidation products;

B. by second oxidation enzymatic activity first oxidation products enzymatic oxygen is changed into second oxidation products;

C. by the 3rd oxidation enzymatic activity second oxidation products enzymatic oxygen changed into the 3rd oxidation products;

D. by a reduction enzymatic activity the 3rd oxidation products enzymatic oxygen changed into 2-KLG.

19. the method for claim 18, at least one needs the enzymatic cofactor of an oxidised form in wherein said first, second and the 3rd the oxidation enzymatic activity, described reduction enzymatic activity needs the described enzymatic cofactor of a reduction form, and the recirculation between at least one oxidation step and this reduction step of the described cofactor of the described cofactor of wherein said oxidised form and described reduction form.

20. the method for claim 19, wherein said first oxidation enzymatic activity needs the described enzymatic cofactor of an oxidised form.

21. the method for claim 18, wherein said carbon source are glucose, described first enzymatic activity is a glucose dehydrogenase activity.

22. the method for claim 21, wherein said glucose dehydrogenase activity can obtain from bacterium, yeast or fungic origin.

23. the method for claim 22, wherein said glucose dehydrogenase activity can obtain from comprising the source of having a liking for acid heat bacterium, first cryptococcus and Bacillaceae bacterial classification.

24. the method for claim 19, wherein said first, described second and described the 3rd enzyme each all be a dehydrogenase activity.

25. the method for claim 19, wherein said first, described second, the described the 3rd and described the 4th enzymatic activity at least a being immobilized.

26. the method for claim 19, wherein said first, described second, the described the 3rd and described the 4th enzymatic activity at least a in dissolved state.

27. the method for claim 25, wherein said second enzyme is a GADH activity.

28. the method for claim 25, wherein said the 3rd enzyme is the KDGDH activity.

29. the method for claim 25, wherein said the 4th enzyme is a reductase activity.

30. the method for claim 29, wherein said reductase activity can obtain from bacterium, yeast or fungic origin.

31. the method for claim 29, wherein said source comprises corynebacterium and erwinia.

32. the method for claim 31, wherein said reductase activity are 2,5 DKG reductase enzymes.

33. the method for claim 18, wherein said first oxidation products is a glyconic acid, and described second oxidation products is 2-KDG, and described the 3rd oxidation products be 2,5-DKG.

34. the method for claim 18, this method is carried out in containing the environment of recombinant host cell.

35. the method for claim 34, wherein said host cell are what live.

36. the method for claim 34, wherein said host cell are nonviable.

37. the method for claim 34, wherein said recombinant host cell comprises the member of enterobacteriaceae.

38. the method for claim 34, this method are carried out in the environment that contains the recombinant host cell film and wherein said first, described second and described the 3rd enzyme at least a and described host cell membrane combine.

39. the method for claim 37, wherein said recombinant host cell are a general Pseudomonas bacterial classification.

40. the method for claim 39, wherein said recombinant host cell are the general bacterium of lemon.

41. the method for claim 40, at least one naturally occurring dehydrogenase activity has sudden change in the wherein said recombinant host cell.

42. the method for claim 41, wherein said sudden change is in membrane-bound GDH activity.

43. the method for claim 41, wherein said host cell also contain the active nucleic acid of coding allos GDH.

44. the method for claim 43, wherein said allos GDH activity can obtain from having a liking for acid heat bacterium, first cryptococcus or Bacillaceae bacterial classification.

45. the method for claim 18, the described enzymatic cofactor of wherein said oxidised form is NADP+, and the described enzymatic cofactor of described reduction form is NADPH.

46. the method for claim 18, the described enzymatic cofactor of wherein said oxidised form is NAD, and described reduction form is NADH.

47. claim 1,15 or 18 method, this method is a continuation method.

48. claim 1,15 or 18 method, this method is a discontinuous method.

49. claim 1,15 or 18 method, this method is carried out in containing the environment of organic solvent.

50. claim 1,15 or 18 method, this method is carried out in containing the environment of long polymkeric substance.

51. claim 14,15 or 18 method also comprise the step that is obtained ASA by described 2-KLG.

52. a host cell contains the nucleic acid that has sudden change in the active gene of coding GHD.

53. a host cell contains the nucleic acid that has sudden change in the active gene of encoded K DGDH.

54. the host cell of claim 52 or 53, this host cell are a general Pseudomonas bacterial classification.

55. the host cell of claim 52 also contains the active nucleic acid of coding allos GDH.

56. the host cell of claim 55 also contains the nucleic acid of coding allos reductase activity.

57. the method for claim 1 randomly comprises the step that reclaims described KDG or DKG.

58. claim 14,15 or 18 method, wherein said 2-KLG is further purified through electrodialysis.

59. the method for claim 45 or 46, wherein said cofactor is through the nanofiltration purifying.

60. the method for claim 18, this method is carried out in containing the environment of salt.

61. the method for claim 60, wherein said salt comprises ammonium sulfate, sodium acetate, ammonium acetate, ammonium chloride, sodium sulfate, potassiumphosphate, sodium phosphate, sodium-chlor, KCl, NH ₄Cl, K ₂SO ₄And NaI.

62. the method for claim 60, wherein salt concn is 0mM-500mM.

Images